Application of Steroid Derivatized Fluorescent Probes and Gold Nanoparticles to Label and Purify Corresponding Binding Proteins
Date Issued
2005
Date
2005
Author(s)
Liang, Ming-Jen
DOI
zh-TW
Abstract
The aims of this thesis are to synthesize fluorescent steroid derivatives, which can be used to label the corresponding steroid binding protein and estrogen receptor in vivo, and to fabricate pregnenolone-capped gold nanoparticles for further purifying the pregnenolone binding protein.
It was shown that pregnenolone can help stabilize the structure of microtubules in zebrafish embryo and affects the cell movement. Presumably there might be pregnenolone binding protein, which can control the formation of microtubules in zebrafish embryo. Motivated by this observation and presumption, we designed and synthesized fluorescent probes to locate the corresponding binding protein. The fluorescent molecular probe features the pregnenolone derivative and a fluorophore, which can be fluorescein or 3-hydroxyflavone, bridged by a hydrocarbon linker. The result showed that the molecular probe incorporated with fluorescein can penetrate into the zebrafish embryo. By utilizing confocal microscope, the pregnenolone binding protein can be located with the aid of fluorescein antibody. The fluorescent probe, which consists of 3-hydroxyflavone derivatives, was also synthesized intending to serve different functions. 3-Hydroxyflavone is very sensitive to the microenvironmental polarity resulting in fluorescent wavelength or quantum yield change. Taking advantage of this unique photoproperties, the probe potentially can detect not only the location of pregnenolone binding protein but monitor its concentration variation in real time. The gold nanoparticles capped with pregnenolone derivative were also fabricated. These modified gold nanoparticles present multi cognate substrates for interacting with the pregnenolone binding protein, rendering the isolation of this protein from the crashed embryos. In combination of MALDI-TOF and polyacrylamide gel electrophoresis, the identity of the isolated protein will be verified.
The estradiol-fluorescein fluorescent molecular probe was also synthesized and applied to study the cellular trafficking of the estradiol. Western blot analysis revealed that the molecular probe indeed can penetrate into cells and bind with its receptor in the cytoplasm. Moreover, the ternary complex can further enter into the nucleus.
Subjects
螢光探針
金奈米粒子
孕烷醇酮
fluorescent probe
gold nanoparticle
pregnenolone
Type
thesis
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